Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.

Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Fecal Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) RNA Is Associated With Decreased Coronavirus Disease 2019 (COVID-19) Survival.

The clinical significance of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA in stool remains uncertain. We found that extrapulmonary dissemination of infection to the gastrointestinal tract, assessed by the presence of SARS-CoV-2 RNA in stool, is associated with decreased coronavirus disease 2019 (COVID-19) survival. Measurement of SARS-CoV-2 RNA in stool may have utility for clinical risk assessment.

Monoclonal antibody therapy for Junin virus infection.

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

Comparative Transcriptomics in Ebola Makona-Infected Ferrets, Nonhuman Primates, and Humans.

The domestic ferret is a uniformly lethal model of infection for 3 species of Ebolavirus known to be pathogenic in humans. Reagents to systematically analyze the ferret host response to infection are lacking; however, the recent publication of a draft ferret genome has opened the potential for transcriptional analysis of ferret models of disease. In this work, we present comparative analysis of longitudinally sampled blood taken from ferrets and nonhuman primates infected with lethal doses of the Makona variant of Zaire ebolavirus. Strong induction of proinflammatory and prothrombotic signaling programs were present in both ferrets and nonhuman primates, and both transcriptomes were similar to previously published datasets of fatal cases of human Ebola virus infection.

In Vitro and In Vivo Activity of Amiodarone Against Ebola Virus.

At the onset of the 2013-2016 epidemic of Ebola virus disease (EVD), no vaccine or antiviral medication was approved for treatment. Therefore, considerable efforts were directed towards the concept of drug repurposing or repositioning. Amiodarone, an approved multi-ion channel blocker for the treatment of cardiac arrhythmia, was reported to inhibit filovirus entry in vitro. Compassionate use of amiodarone in EVD patients indicated a possible survival benefit. In support of further clinical testing, we confirmed anti-Ebola virus activity of amiodarone in different cell types. Despite promising in vitro results, amiodarone failed to protect guinea pigs from a lethal dose of Ebola virus.

Fully Human Immunoglobulin G From Transchromosomic Bovines Treats Nonhuman Primates Infected With Ebola Virus Makona Isolate.

Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.

Interferon-ß and Interferon-γ Are Weak Inhibitors of Ebola Virus in Cell-Based Assays.

Previous studies have demonstrated little efficacy of interferons (IFNs) in animal models of Ebola virus disease. However, these studies were limited to a small number of type I IFNs and, during the most recent outbreak of Ebola virus, questions regarding the suitability of the animal models to evaluate IFNs were raised. To address the potential that anti-Ebola virus activity was overlooked, type I and type II IFNs (α-2a, α-2b, -ß, -γ, and -universal) were tested in a variety of cell types (Vero E6, Huh 7 cells, and human macrophages). IFNs are weak inhibitors of Ebola virus Makona in these cell lines.

Necrotizing Scleritis, Conjunctivitis, and Other Pathologic Findings in the Left Eye and Brain of an Ebola Virus-Infected Rhesus Macaque (Macaca mulatta) With Apparent Recovery and a Delayed Time of Death.

A 3.5-year-old adult female rhesus macaque (Macaca mulatta) manifested swelling of the left upper eyelid and conjunctiva and a decline in clinical condition 18 days following intramuscular challenge with Ebola virus (EBOV; Kikwit-1995), after apparent clinical recovery. Histologic lesions with strong EBOV antigen staining were noted in the left eye (scleritis, conjunctivitis, and peri-optic neuritis), brain (choriomeningoencephalitis), stomach, proximal duodenum, and pancreas. Spleen, liver, and adrenal glands, common targets for acute infection, appeared histologically normal with no evidence of EBOV immunoreactivity. These findings may provide important insight for understanding sequelae seen in West African survivors of Ebola virus disease.

In vivo Ebola virus infection leads to a strong innate response in circulating immune cells.

BACKGROUND: Ebola virus is the causative agent of a severe syndrome in humans with a fatality rate that can approach 90 %. During infection, the host immune response is thought to become dysregulated, but the mechanisms through which this happens are not entirely understood. In this study, we analyze RNA sequencing data to determine the host response to Ebola virus infection in circulating immune cells. RESULTS: Approximately half of the 100 genes with the strongest early increases in expression were interferon-stimulated genes, such as ISG15, OAS1, IFIT2, HERC5, MX1 and DHX58. Other highly upregulated genes included cytokines CXCL11, CCL7, IL2RA, IL2R1, IL15RA, and CSF2RB, which have not been previously reported to change during Ebola virus infection. Comparing this response in two different models of exposure (intramuscular and aerosol) revealed a similar signature of infection. The strong innate response in the aerosol model was seen not only in circulating cells, but also in primary and secondary target tissues. Conversely, the innate immune response of vaccinated macaques was almost non-existent. This suggests that the innate response is a major aspect of the cellular response to Ebola virus infection in multiple tissues. CONCLUSIONS: Ebola virus causes a severe infection in humans that is associated with high mortality. The host immune response to virus infection is thought to be an important aspect leading to severe pathology, but the components of this overactive response are not well characterized. Here, we analyzed how circulating immune cells respond to the virus and found that there is a strong innate response dependent on active virus replication. This finding is in stark contrast to in vitro evidence showing a suppression of innate immune signaling, and it suggests that the strong innate response we observe in infected animals may be an important contributor to pathogenesis.

Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.

Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques.

UNLABELLED: Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model. IMPORTANCE: In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

Comparison of respiratory inductive plethysmography versus head-out plethysmography for anesthetized nonhuman primates in an animal biosafety level 4 facility.

For inhalational studies and aerosol exposures to viruses, head-out plethysmography acquisition has been traditionally used for the determination of estimated inhaled dose in anesthetized nonhuman primates prior to or during an aerosol exposure. A pressure drop across a pneumotachograph is measured within a sealed chamber during inspiration/exhalation of the nonhuman primate, generating respiratory values and breathing frequencies. Due to the fluctuation of depth of anesthesia, pre-exposure respiratory values can be variable, leading to less precise and accurate dosing calculations downstream. Although an anesthesia infusion pump may help stabilize the depth of sedation, pumps are difficult to use within a sealed head-out plethysmography chamber. Real-time, head-out plethysmography acquisition could increase precision and accuracy of the measurements, but the bulky equipment needed for head-out plethysmography precludes real-time use inside a Class III biological safety cabinet, where most aerosol exposures occur. However, the respiratory inductive plethysmography (RIP) acquisition method measures the same respiratory parameters by detecting movement of the chest and abdomen during breathing using two elastic bands within the Class III biological safety cabinet. As respiratory values are relayed to a computer for software integration and analysis real-time, adjustment of aerosol exposure duration is based on the depth of sedation of the animal. The objective of this study was to compare values obtained using two methodologies (pre-exposure head-out plethysmography and real-time RIP). Transitioning to RIP technology with real-time acquisition provides more consistent, precise, and accurate aerosol dosing by reducing reported errors in respiratory values from anesthesia variability when using pre-exposure head-out plethysmography acquisition.

Optimized microRNA purification from TRIzol-treated plasma.

BACKGROUND: MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route. RESULTS: Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 µL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 µg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling. CONCLUSIONS: This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.

Lassa and Marburg viruses elicit distinct host transcriptional responses early after infection.

BACKGROUND: Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosis is difficult because patients infected with either pathogen present similar nonspecific symptoms early after infection. Current diagnostic tests are based on detecting viral proteins or nucleic acids in the blood, but these cannot be found during the early stages of disease, before the virus starts replicating in the blood. Using the transcriptional response of the host during infection can lead to earlier diagnoses compared to those of traditional methods. RESULTS: In this study, we use RNA sequencing to obtain a high-resolution view of the in vivo transcriptional dynamics of peripheral blood mononuclear cells (PBMCs) throughout both types of infection. We report a subset of host mRNAs, including heat-shock proteins like HSPA1B, immunoglobulins like IGJ, and cell adhesion molecules like SIGLEC1, whose differences in expression are strong enough to distinguish Lassa infection from Marburg infection in non-human primates. We have validated these infection-specific expression differences by using microarrays on a larger set of samples, and by quantifying the expression of individual genes using RT-PCR. CONCLUSIONS: These results suggest that host transcriptional signatures are correlated with specific viral infections, and that they can be used to identify highly pathogenic viruses during the early stages of disease, before standard detection methods become effective.

Virus nomenclature below the species level: a standardized nomenclature for filovirus strains and variants rescued from cDNA.

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to "Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1" (with the suffix "rec" identifying the recombinant nature of the virus and "abc1" being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as "EBOV H.sap/COD/95/Kik-abc1") and abbreviations (such as "EBOV/Kik-abc1") could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.

Low-Input, High-Resolution 5' Terminal Filovirus RNA Sequencing with ViBE-Seq.

Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5' and 3' ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5' end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5' end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.

A Single-shot ChAd3 Vaccine Provides Protection from Intramuscular and Aerosol Sudan Virus Exposure.

Infection with Sudan virus (SUDV) is characterized by an aggressive disease course with case fatality rates between 40-100% and no approved vaccines or therapeutics. SUDV causes sporadic outbreaks in sub-Saharan Africa, including a recent outbreak in Uganda which has resulted in over 100 confirmed cases in one month. Prior vaccine and therapeutic efforts have historically prioritized Ebola virus (EBOV), leading to a significant gap in available treatments. Two vaccines, Erbevo ® and Zabdeno ® /Mvabea ® , are licensed for use against EBOV but are ineffective against SUDV. Recombinant adenovirus vector vaccines have been shown to be safe and effective against filoviruses, but efficacy depends on having low seroprevalence to the vector in the target human population. For this reason, and because of an excellent safety and immunogenicity profile, ChAd3 was selected as a superior vaccine vector. Here, a ChAd3 vaccine expressing the SUDV glycoprotein (GP) was evaluated for immunogenicity and efficacy in nonhuman primates. We demonstrate that a single dose of ChAd3-SUDV confers acute and durable protection against lethal SUDV challenge with a strong correlation between the SUDV GP-specific antibody titers and survival outcome. Additionally, we show that a bivalent ChAd3 vaccine encoding the GP from both EBOV and SUDV protects against both parenteral and aerosol lethal SUDV challenge. Our data indicate that the ChAd3-SUDV vaccine is a suitable candidate for a prophylactic vaccination strategy in regions at high risk of filovirus outbreaks. One Sentence Summary: A single-dose of ChAd3 vaccine protected macaques from lethal challenge with Sudan virus (SUDV) by parenteral and aerosol routes of exposure.

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever.

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.

A broad-spectrum antiviral targeting entry of enveloped viruses.

We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus-cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure-activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus-cell but not cell-cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus-cell fusion.